1. Field of the Invention:
This invention is directed to a method of separation and extraction of organic compounds, inorganic compounds, biomolecules and biomaterials by partition using an aqueous two-phase solvent system.
2. Discussion of the Background:
The separation of a mixture by distribution between two immiscible liquids, either by bulk extraction or by liquid-liquid partition chromatography is known. Especially well known in this regard are systems containing immiscible organic solvents and water. Also, advantage has been taken of the phase separation that frequently occurs when solutions of two structually different water-soluble polymers are mixed above critical concentrations. These systems spontaneously separate into two immiscible liquid phases, each phase enriched with respect to one of the polymers. Such aqueous two-phase systems are suitable for separation of labile materials such as enzymes, cells and organelles. Aqueous phase systems containing two polymers, most commonly polyethylene glycol (PEG) and dextran have found wide application for the separation of biological materials. The phases have low osmotic pressure and high water content. Salts and other solutes can be included to provide buffering capacity. Systems containing a single polymer and a high concentration of some particular salt, e.g., PEG and a phosphate, have also proved useful in the separation of macromolecules. However, these systems have not been suitable for the separation of optical isomers. A description of two-polymer systems and polymer-salt systems as applied to separation of biological materials can be found in Walter, H. et al, "Partitioning in Aqueous Two-Phase Systems" (Academic Press, 1985) and Albertsson, P. et al "Partition of Cell Particles and Macromolecules" (Wiley, 1986).
Existing methods to separate optical isomers are based on chromatographic techniques, selective enzymatic reactions, and fractional crystallization of diastereomeric complexes formed with chiral resolving agents. The most common technique used on an industrial scale is fractional crystallization. For example Manghisi et al, U.S. Pat. No. 4,533,748 teaches the use of L-lysine to form diastereomeric salts with a racemic propionic acid derivative followed by fractional crystallization. Fahnenstich et al, U.S. Pat. No. 3,980,665, discloses the use of L-lysine to convert D,L-penicillamine to D-penicillamine. Chibata et al, U.S. Pat. No. 4,519,955, discloses a method of optical resolution of .alpha.-amino acids and .alpha.-phenylethane sulfonic acids by fractional crystallization. Optical resolution of a D,L-amino acid is disclosed in Yukawa et al, U.S. Pat. No. 4,610,820, which comprises reacting the mixture with an optically active N-acylaspartic acid, followed by fractional crystallization.
A method to resolve stereoisomers which relies on a two-phase solvent system is disclosed by Empie, U.S. Pat. No. 4,636,470. The method relies upon the preferential enzymatic hydrolysis of one enantiomer of D,L-phenylalanine. The racemate is dissolved in a substantially water immissible organic material which is a solvent for the amino acid racemate but not for the resolved amino acid.
McCloud (Dissertation Abstract B 1969, 29 (7), 2357-8) discloses the resolution of D,L-camphoric acid, D,L-dibromobutanediol, and D,L-isohydrobenzolin isomers by solvent extraction using water and D-tartrate esters in a two-phase system.
A method of separation of optical isomers based on stereospecific interactions with asymmetric sorbents and solvents was disclosed by Buss et al, Ind. Eng. Chem., V60 (8), p. 12-28, 1968, however asymmetric solvents are prohibitively expensive. The extensive reviews of chiral adsorbents for analytical separation of optical isomers can be found in Lough, W. J. et al, "Chiral Liquid Chromatography" (Blackie and Son, 1989) and Allenmark, S.G., "Chromatographic Enantioseparation: Methods and Applications" (Ellis Horwood Limited, 1988).
These methods to separate optical isomers are expensive and require several steps. They are highly specialized procedures which must be developed specifically for the optical isomer mixture to be resolved. For example, conditions which effect the selective crystallization of a diastereomeric mixture must be carefully controlled with respect to concentration and mass transfer effects. In addition, these systems often do not yield the high purity product one desires.
Therefore a need still exists for inexpensive and generally applicable methods for separating stereoisomers and biomolecules especially as applied to industrial processes.